Yahiaoui B, Loyens M, Taibi A, Schöneck R, Dubremetz J F, Ouaissi M A
Research Laboratory on Trypanosomatids, INSERM U 167, Institut Pasteur, Lille, France.
Parasitology. 1993 Dec;107 ( Pt 5):497-507. doi: 10.1017/s0031182000068074.
In the present study we have used antibodies to Leishmania major promastigote antigens which were eluted from a glutathione-agarose column (LmGbp) and could identify several parasite components among different Leishmania species by using immunoprecipitation and Western blot techniques. The results also showed that some of LmGbp are present among the molecules released into the culture medium. Moreover, immunofluorescence assays clearly demonstrated that LmGbp are expressed by intracellular amastigotes. The electron micrographs of thawed cryosections of L. major-infected cells revealed that the antigens were associated with the membrane of the phagocytic vacuole. Moreover, the Western blot technique allowed us to identify, using other Leishmania species extracts and anti-LmGbp antibodies, a major polypeptide of an apparent molecular mass of 66 kDa. Immunofluorescence studies suggested that the 66 kDa polypeptide is associated with intracytoplasmic vesicles. Cryosections of Leishmania promastigotes improved the fine structure preservation of the organelles and enabled a number of features to be seen, particularly the structures considered as vesicles, which appeared as a complex tubulo-vesicular structure resembling mammalian cell endosomes and Leishmania organelles previously named 'megasomes'. Further studies using antibodies against the native 66 kDa protein will be needed to investigate the localization of the protein at the ultrastructural level and to follow its intracellular vesicular traffic.
在本研究中，我们使用了针对从谷胱甘肽琼脂糖柱洗脱的利什曼原虫前鞭毛体抗原的抗体（LmGbp），通过免疫沉淀和蛋白质印迹技术，该抗体能够识别不同利什曼原虫物种中的几种寄生虫成分。结果还表明，一些LmGbp存在于释放到培养基中的分子中。此外，免疫荧光分析清楚地表明，LmGbp由细胞内无鞭毛体表达。大利什曼原虫感染细胞的解冻冷冻切片的电子显微照片显示，抗原与吞噬泡膜相关。此外，蛋白质印迹技术使我们能够使用其他利什曼原虫物种提取物和抗LmGbp抗体鉴定出一种表观分子量为66 kDa的主要多肽。免疫荧光研究表明，66 kDa多肽与胞质内小泡相关。利什曼原虫前鞭毛体的冷冻切片改善了细胞器的精细结构保存，并使许多特征得以显现，特别是被认为是小泡的结构，其呈现为类似于哺乳动物细胞内体和先前命名为“大滋养体”的利什曼原虫细胞器的复杂管状小泡结构。需要使用针对天然66 kDa蛋白的抗体进行进一步研究，以在超微结构水平上研究该蛋白的定位，并追踪其细胞内小泡运输。
